Cell Biology

Interfering with the ability of telomerase to bind to its telomeric substrate provides a potential avenue for telomerase inhibition, through either direct inhibition of the enzyme, or indirectly through sequestration of its telomeric substrate. Telomeric DNA, owing to its repetitive G-rich sequence (TTAGGG), has the ability to form a compact folded structure called a G-quadruplex, wherein four guanine bases use Hoogsteen hydrogen bonding to form a stable square-planar arrangement.

G-quadruplexes were long thought to block extension by telomerase through sequestration of the single-stranded 3′-end. However, research in the CBU has revealed that a particular conformational subset of G-quadruplexes are indeed utilised as a substrate by human and ciliated protozoan telomerase (Oganesian et al., EMBO J 2006; Oganesian et al., Biochemistry 2007; Moye, Porter, Cohen, et al., Nature Communications 2015).

We collaborated with the laboratory of Professor Antoine van Oijen at the University of Wollongong to probe the mechanism of telomerase extension of G-quadruplexes using single-molecule fluorescence resonance energy transfer (smFRET) microscopy (Paudel, Moye et al., eLife 2020). Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

We work extensively with small-molecule ligands that specifically stabilise G-quadruplexes. In collaboration with Dr Jennifer Beck in the School of Chemistry at the University of Wollongong, we aim to evaluate ligands that may be specific for a particular conformation of G-quadruplex. Such compounds are also a valuable tool for looking at G-quadruplexes at the cellular level. Our guiding hypothesis is that different conformations of G-quadruplex will perform distinct functions at the telomere.

Telomerase is a multi-subunit protein-RNA complex. The way in which these subunits are assembled into an active enzyme complex and then transported (“recruited”) to the telomere is complex and not fully understood. We have established in our laboratory a method to quantitatively detect telomerase at the telomere in human cells - not an easy task due to its extreme rarity, even in cancer cells. We have optimised the technique of Fluorescence in situ hybridisation (FISH) using a probe complementary to the telomeric DNA and concomitant FISH against hTR; colocalisation of the two probes indicates telomerase at the telomere.

We found that small structures within the cell nucleus called Cajal Bodies are an important stop-over point for telomerase on its way to the telomere. A protein called TCAB1 was known to transport telomerase to Cajal Bodies; we demonstrated that TCAB1 plays an additional role in recruitment by accompanying telomerase all the way to the telomere (Stern et al., Mol. Cell. Biol. 2012).

More recently, we made the surprising discovery that the cellular response to DNA damage is tightly linked to the process of telomerase recruitment to telomeres in human cells. We demonstrated that the DNA damage response kinases ATM and ATR are required for the presence of telomerase at telomeres, and that DNA replication fork stalling triggers ATR-mediated telomerase recruitment (Tong, Stern et al., Cell Reports 2015). This implies that telomeres exploit DNA damage signalling pathways to regulate telomerase presence at the telomere. Furthermore, telomerase and the DNA damage response are both key drivers of oncogenesis, and our data demonstrate that they are tightly linked. We are continuing to elucidate the mechanistic details of the involvement of DNA damage response pathways in bringing telomerase to the telomere.

A subset of inherited bone marrow failure (BMF) syndromes is caused by mutations in genes encoding telomere-related proteins, including hTERT and hTR. In these patients, telomeres become abnormally short, leading to BMF disorders known collectively as Telomere Biology Disorders (TBDs), including Dyskeratosis Congenita (DC). The causative gene remains unidentified in ~30-40% of DC patients, and our team leads novel gene discovery efforts in Australian TBD patients. Disease-associated mutation in the gene ACD, encoding the telomere-binding protein TPP1, was first described by us in an Australian patient (Guo et al., Blood 2014), and independently identified in a family in the USA (Kocak et al., Genes & Dev. 2014).

We are continuing to apply our expertise in telomerase biology to functionally characterise the impact of newly-identified patient mutations on telomerase biogenesis, trafficking and activity. Our laboratory has established a variety of biochemical assays, such as measuring the affinity K(d) between telomerase and its DNA substrate; the stability k(off) of the enzyme-DNA complex; and activity using a direct (non-PCR) assay (Cohen & Reddel, Nature Methods 2008; Tomlinson, Cohen & Bryan, Methods 2017).

These assays have been enabled by our established system of enzyme over-expression and purification, and have provided new insights into the complex interplay between substrate association/dissociation and conformational change(s) throughout the nucleotide addition cycle. For example, such enzymatic assays led to the conclusion that the region of hTERT known as the TEN domain contributes high-affinity binding to the DNA substrate, and also functions in positioning the 3′ end of the DNA in the active site (Jurczyluk et al., Nucleic Acids Res. 2010). We found that human telomerase binds its DNA substrate using a two-step mechanism involving active-site conformational changes (Tomlinson et al., Biochem. J. 2015), and we were the first to demonstrate that the C-terminus of hTERT provides high-affinity binding to the DNA substrate (Tomlinson et al., Biochimie 2016).

Our characterisation of the biochemical defect caused by newly-identified telomerase mutations in Australian bone marrow failure patients is enabling definitive molecular diagnoses for families carrying such mutations, and in some cases impacting on treatment decisions for the patients.

Elucidating a high-resolution 3-D molecular structure of the human telomerase enzyme complex is being led by Dr Scott Cohen. In biochemistry and enzymology, structure dictates function. Detailed knowledge of an enzyme’s structure can reveal its mechanism of action, provide an understanding of how mutations can cause disease, and facilitate the rational design of small-molecule inhibitors with therapeutic potential.

Dr Cohen is using electron microscopy to provide structural information. The image below shows individual molecules of highly purified telomerase, stained to enhance contrast. Close examination reveals that the molecules consist of two lobes connected by a narrow hinge, consistent with the conclusion of his 2007 Science paper.

Current efforts are directed towards obtaining high-resolution data using cryogenic electron microscopy (cryo-EM), wherein molecules are captured within a thin aqueous glass cooled in liquid nitrogen. Cryo-EM provides high-resolution images of molecules in their native state as they exist in solution and can also reveal conformational changes the enzyme may undergo.

The long-term aim of the lab’s research is to apply the knowledge of structure to the rational and targeted design of small-molecule telomerase inhibitors as potential cancer therapeutics.